Large-scale next generation sequencing based analysis of SLCO1B1 pharmacogenetics variants in the Saudi population

Background SLCO1B1 plays an important role in mediating hepatic clearance of many different drugs including statins, angiotensin-converting enzyme inhibitors, chemotherapeutic agents and antibiotics. Several variants in SLCO1B1 have been shown to have a clinically significant impact, in relation to efficacy of these medications. This study provides a comprehensive overview of SLCO1B1 variation in Saudi individuals, one of the largest Arab populations in the Middle East. Methods The dataset of 11,889 (9,961 exomes and 1,928 pharmacogenetic gene panel) Saudi nationals, was used to determine the presence and frequencies of SLCO1B1 variants, as described by the Clinical Pharmacogenetic Implementation Consortium (CPIC). Results We identified 141 previously described SNPs, of which rs2306283 (50%) and rs4149056 (28%), were the most common. In addition, we observed six alleles [*15 (24.7%) followed by *20 (8.04%), *14 (5.86%), *5 (3.84%), *31 (0.21%) and *9 (0.03%)] predicted to be clinically actionable. Allele diplotype to phenotype conversion revealed 41 OATP1B1 diplotypes. We estimated the burden of rare, and novel predicted deleterious variants, resulting from 17 such alterations. Conclusions The data we present, from one of the largest Arab cohorts studied to date, provides the most comprehensive overview of SLCO1B1 variants, and the subsequent OATP1B1 activity of this ethnic group, which thus far remains relatively underrepresented in available international genomic databases. We believe that the presented data provides a basis for further clinical investigations and the application of personalized statin drug therapy guidance in Arabs. Supplementary Information The online version contains supplementary material available at 10.1186/s40246-024-00594-9.


Background
Pharmacogenomics (PGx) has made important advances in recent years and is now being rapidly translated to clinical care.Organic anion transporting polypeptide (OATP) transporters, encoded by the SLCO gene family, are involved in absorption, distribution, metabolism and excretion of many drugs.They are crucial in drug transport across the cellular intestinal membranes, renal and pulmonary epithelial cells, brain capillary endothelial cells, hepatocytes and cancer cells [1,2].SLCO1B1 plays an important role in mediating hepatic clearance of many different drugs including statins such as simvastatin, angiotensin-converting enzyme inhibitors such as enalapril, chemotherapeutic agents, antibiotics such as rifampicin, the endothelin receptor antagonist besentan, the prostacyclin beraprost, the anti-inflammatory drug diclofenac, and the antiviral simeprevir [3,4].
Genetic variations can have a profound impact on how an individual responds to drugs.Knowledge of such variation in a patient may facilitate better therapeutic options leading to better outcomes, and in some instances, preventing adverse drug events (ADE's).For instance, simvastatin which is commonly used in the treatment of hypercholesterolemia, one of the most significant risk factors in cardiovascular disease, may lead to statininduced myopathy (SIM) [5,6].Clinical trials observed that some patients on statins achieved similar benefits at lower doses when compared to others [7,8].The differences in drug efficacy highlight the current trend towards individualized pharmacotherapy, such that the right drug is delivered at the right dose for the right patient.A standard dose of a given drug is not always safe, effective or economical in an individual patient.
Allelic frequencies of genes encoding drug-metabolizing enzymes including SLCO1B1 may vary considerably between ethnic groups, with incidence of SLCO1B1*5, *15 and *17 haplotypes being well documented in Caucasians and in some other ethnicities [11][12][13], but to date, poorly in Arabs.In this regard, this study will provide new pharmacogenetic information, specific for one of the largest Arab populations in the Middle East.Identification of known and novel allele frequencies in the Saudi population, and their phenotypic designation, will provide the basis for better clinical management of hypercholesterolemia, in Saudi Arabia and beyond.
The frequency of the most common diplotype *1/15 (21.32%) is similar to that in two other populations (Americans and Near Eastern).Among high frequency diplotypes, *1/37 is much less commonin Saudi individuals relative to American, European, Asians, Near Eastern and African populations (p < 0.05).Diplotype *1/14 (5.50%) has been previously reported in the European population and no other ethnic groups (Table S3).Predicted actionable phenotypes (poor function: 9.5% and decreased function: 38.1%) were observed in 47.6% of the Saudi population.SLCO1B1 phenotypes and their frequencies in comparison to other ethnicities are based upon CPIC data as presented in Table 2 (https://cpicpgx.org/guidelines/cpic-guideline-for-statins/).
Diplotypes associated with an increased function phenotype were identified in 2.5% of our study population (Fig. 2, Table S4).We estimated the burden of rare and novel predicted deleterious variants, to have an aggregated frequency of 2.3% resulting from 17 alterations.They included 1 stop-gain, 4 splice donor, and 12 missense variants, with an ADME score calculated as described in the methods, of ≥ 80% (Table 3).
The potential for genotype-based prescription of statins is indicated by historical data from King Faisal Specialist Hospital and Research Centre (KFSHRC).At KFSHRC,from 2016 to 2019, a total of 273,167 statin drugs (simvastatin and atorvastatin) had been received by 64,034 patients.We identified three groups of patients: those who had received only simvastatin, only atorvastatin and both statins (Table 4).Proportions of simvastatin and atorvastatin prescribed for patients at KFSHRC changed between 2016 and 2019.In 2016 simvastatin was more frequently prescribed when compared to atorvastatin (7534 vs.7436) whereas from 2017 this trend had changed and atorvastatin became the more frequent medication than simvastatin with a ratio of ~ 2:1 in 2019 (Fig. 3).While statin induced toxicity may largely be resolved at KFSHRC, genotype-based statin prescription may be beneficial in the larger Saudi population.

Discussion
Variants of the SLCO1B1 transporter impact the hepatic uptake of statins and a large number of endogenous substrates.The most common statin-related adverse drug reaction (ADR) is skeletal muscle toxicity that results in statin-associated musculoskeletal symptoms (SAMS) [14,15].In 2008, data from a big genome-wide study showed a strong association between a SLCO1B1 variant (rs4149056) and an increased risk of statin-induced myopathy.This finding was further confirmed by additional studies that revealed an odds ratio (OR) of 2.3 and 3.2, for myopathy in subjects exposed to simvastatin, with the CT and CC genotypes of rs4149056, respectively [16].This finding has since been replicated in an independent trial and practice-based longitudinal cohort of 20,000 subjects [17].In 2012, the Clinical Pharmacogenetics Implementation Consortium (CPIC) released gene-based prescribing guidelines for simvastatin based on SLCO1B1 genotypes [18], updated further in 2014 [10] and again in 2022 [19].They provided therapeutic recommendations for statins with the goal of improving the overall safety, adherence and effectiveness of statin therapy.Individualized pharmacotherapy using the right drug and at the right dose is very critical to optimal outcomes in patients on statins.Allele frequencies of SLCO1B1 vary across multiple ancestries and geographically diverse groups (https://cpicpgx.org/guidelines/cpicguideline-for-statins/).To the best of our knowledge, this is the first study of SLCO1B1 polymorphism in the population of Saudi Arabia, a population not well represented in current databases.Mining of large-scale NGS data is a very powerful tool for cataloguing the range and frequency of genetic variation in populations [20].Many SNPs have been identified in SLCO1B1 but thus far, only a few are known to have functional effects [14,21].We found that the common C allele at (c.521T > C) of rs4149056 (SLCO1B1 *5) and the SLCO1B1 *15 alleles associated with lower plasma clearance of simvastatin was present with higher frequency in the Saudi population when compared to other ethnic groups (Table S2).The high frequency of the c.521T > C allele (28%) results in the higher frequency of *allele diplotypes based on which SLCO1B1 phenotypes are assigned.
Predicted actionable diplotypes (decreased and poor function), that are associated with an increased or high risk of myopathy were strongly represented in our study population.Their frequency was higher than seen in another Middle East population from Qatar (47.61% versus 32.37% respectively).It was also much higher compared to European, East Asian, Near Eastern and American populations (~ 29%), and thus far, the highest observed in the CPIC database (Table S4).We can estimate that ~ 12,000 (47.61%) patients who received simvastatin at KFSHRC from 2016 to 2019 are      Our study, as well as data from other Middle East populations [22], suggest that Arabs may be at greater risk of simvastatin induced myopathy.Since there are alternate hypolipidemic drugs, that have a lower substrate affinity for OATP1B, they may be alternatives for patients intolerant of statins.Implementation of SLCO1B1genotyping before statins are prescribed [19], could be very helpful in Arabs.The current clinical prediction of SLCO1B1 phenotypes is developed based on studies conducted mainly in Europeans and Americans.
To the best of our knowledge, such studies have never been performed in Arabs.It is well known that simvastatin-related muscle toxicity still occurs in patients with WT alleles of rs4149056 and we cannot thus exclude the possibility of other potentially deleterious variants in SLCO1B1 closely linked to rs4149056 [18].The high number of individuals who carry actionable SLCO1B1 dilpotypes, strongly support further studies in Middle Eastern populations.Individuals who have developed clinically and laboratory confirmed SAMS, should be further investigated to identify novel SLCO1B1 variants, that have clinical significance.Such studies in the Arab population have substantial potential to discover new variants that underlie statin-related myopathy.By leveraging large-scale whole-exome sequencing data from Saudi individuals, we for the first time, present data on novel, and predicted deleterious SLCO1B1 variants (Table S1 and Table S5) in Arabs.At present, pharmacogenetic testing is performed mainly by SNP arrays targeting specific alleles, and hence, the detection of rare and novel variants is not possible.Unexplained statin toxicity should not be neglected and should be studied further, as rare or novel variants may account for nearly all inter-individual variabilities in pharmacogenes [23].Rare deleterious variants with an aggregated frequency of 1.2%, jointly explained 8.7% of the genetic basis of SLCO1B1 variability in other studies [24].Considering the high frequency (2.3%), of rare and novel predicted pathogenic alterations, detected by this study in the Saudi population, it is important to determine their clinical impact.Notably, the nonsense c.1706 C > A:p.S569X with a homozygous frequency of 0.03% should be further investigated.

Conclusions
Our study highlights the value of mining large NGS databases, as being a powerful tool, to improve knowledge of genomic variation in clinically relevant genes.The data we present from one of the largest Middle Eastern countries, provides the most comprehensive overview of SLCO1B1 variants, and the subsequent OATP1B1 activity in a large cohort of Arabs, a population still underrepresented in available international genomic databases.We believe studies such as this, provide important information for the guidance of personalized drug therapies, in Arab patients.

Methods
The dataset used for mining of SLCO1B1 variants comprised 9,961 exomes and 1,928 PGx custom gene panels  S1), from unrelated Arab individuals, sequenced by the Saudi Human Genome Program (SHGP) between 2015 and 2019, as part of a comprehensive investigation of rare diseases in the Saudi population [25,26].The Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines (https://cpicpgx.org/guidelines/)for SLCO1B1 and simvastatin-induced myopathy were used to assign the likely OATP1B1 phenotypes: normal function, decreased function, poor function, increased function, indeterminate function and *allele nomenclature [10].We used Stargazer software (v.1.0.8.5) to compute star alleles.This algorithm performs statistical haplotype phasing using Beagle [27] with reference samples from the 1000 Genomes Project [28].The Beagle method is based on a localized haplotype-cluster model, which is an empirical linkage disequilibrium model, that can take the local structure of data into consideration.The Beagle algorithm is accurate and fast due to the use of an EM-based algorithm that literately fits the best model to the data [27].Afterwards, the phased haplotypes computed by Beagle are matched to publicly available star allele information, mostly in the PharmVar database (https://www.pharmvar.org)and PharmGKB (https://www.pharmgkb.org/).Finally, Stargazer reports the star allele findings in a tabular format, along with prediction of the related metabolizer information.

Fig. 2
Fig. 2 Pie chart presenting the percentage of SLCO1B1 phenotypes found in the Saudi population

Table 3
Novel and rare SLCO1B1 variants in Saudi population

Table 4
Number of patients and prescriptions for simvastatin and atorvastatin at KFSHRC in 2016-2019